Reaction chambers containing complex stable reagent formulations and test kit for detection and isolation of pathogenic microbial nucleic acids

ABSTRACT

The invention relates to standard reaction areas containing complex, storage-stable reagent formulations and thus suited to acceptance and, if need be, storage of complex liquid patient samples, thereafter to be examined for the existence of pathogenic nucleic acids (bacteria, viruses etc.). Preferably, the reaction areas manifest complex, storage-stable reagent formulations permitting detection and quantitative isolation of viral and bacterial nucleic acids in a test kit, even in the existence of extremely low copy numbers from a complex biological sample. At the same time, a simple archiving system for the clinically relevant nucleic acids can be provided. Thanks to the use of said reaction areas containing the complex, storage-stable reagent formulations, necessary process steps of sample handling are drastically reduced and thus potential risks of infection and risks of contamination distinctly lowered.

The invention relates to standard reaction areas containing complex,storage-stable reagent formulations and thus suited to accept and, ifneed be, store complex fluid patient samples, thereafter to be examinedfor the existence of pathogenic nucleic acids (bacteria, viruses etc.).Preferably, the reaction areas manifest complex, storage-stable reagentformulations permitting detection and quantitative isolation of viraland bacterial nucleic acids in a test kit, even in the existence ofextremely low copy numbers from a complex biological sample. At the sametime, a simple archiving system for the clinically relevant nucleicacids can be provided. Thanks to the use of said reaction areascontaining the complex, storage-stable reagent formulations, necessaryprocess steps of sample handling are drastically reduced and thuspotential risks of infection and risks of contamination distinctlylowered.

Screening of complex biological samples (serum, plasma, blood etc.) forthe existence of infectious components is gaining more and moreimportance. Virus infections such as HIV, HCV or HBV are becoming moreand more widespread all over the world, New test methods on the basis ofthe use of sensitive amplification techniques such as PCR or NASBAenable a highly efficient detection of viruses and are being used moreand more frequently as diagnostic instruments. Specialists know that anessential step for the application of these techniques for the detectionof pathogenic nucleic acids in the isolation of the nucleic acids (RNAor DNA) comprises relevant complex clinical samples. Without such ahighly efficient isolation, e.g. of viral nucleic acids, no sufficientlysensitive diagnosis can be carried out.

At the present, the isolation, e.g. of viral nucleic acids from bloodproducts, is customarily done via the lysis of the original materialwith a buffer containing chaotropic components of a high ion strengthand the subsequent binding of the nucleic acids to a solid phase (e.g.membrane filter). The bound nucleic acids are washed on the solid phaseand finally dissolved from the solid phase with a buffer of a suitableion strength.

Inter alia, the process is portrayed in patent U.S. Pat. No. 5,234,809 Aand known world-wide under the name of “Boom Patent”. The processprecisely describes the isolation of nucleic acids from originalmaterials containing the latter by incubation of the original materialwith a chaotropic buffer and a solid phase binding DNA. The chaotropicbuffers implement both the lysis of the original materials and also thebinding of the nucleic acids to the solid phase. The method is wellsuited to isolating nucleic acids from small sample quantities and isapplied in practice specifically in the area of the isolation of viralnucleic acids.

One main problem in the isolation of viral and bacterial nucleic acidsis the realisation of a sufficiently high diagnostic sensitivity, as thenumber of viral copies (or the copy number of other microbial pathogens)in a complex biological sample is very low as a rule. Solutions toincrease the extraction efficiency up to now relate to the increase ofthe volume of the clinical sample to be examined or the enrichment ofviral particles by centrifugation techniques. It becomes clear tospecialists that narrow borders are set for these variants.

An increase of the initial volume of the clinical sample with theextraction methods currently in use leads to a necessary proportional oralso over-proportional increase of the necessary extraction buffers.This subsequently results in a multiplication of necessarycentrifugation steps in order to transfer the increased volume of thesample to be processed to a centrifugation filter on which the bindingof the nucleic acid to be bound is to take place in the end.

In this way, the manual efforts for the extraction, the extraction timeand the contamination risk are increased.

A further problem in the isolation, in particular of viral nucleicacids, for a subsequent diagnostic detection is that a series ofreaction components has to be pipetted into a reaction vessel containingthe sample for the extraction of the nucleic acids.

The task of the invention in question was thus to look for possibilitiesremoving all the existing problems in connection with the handling of acomplex biological sample to be investigated for the existence ofmicrobial nucleic acids and to find reaction approaches permitting asimplification of the methods for the isolation of microbial nucleicacids from patients' samples and further automating the methods, inorder to enable a parallel processing of samples at high throughflow.Further, known risks of cross-contaminations are to be distinctlyreduced with these reaction approaches via the reduction of workingsteps.

The invention is realised by claims 1, 8, 11 and 12, the sub-claimsbeing preferential variants.

The task was solved by standard reaction vessels (such as 1.5 ml or 2.0ml Eppendorf reaction vessels or 96-well or 386-well micro titre plates)containing all the components suited to accept and, if need be, storecomplex fluid patient samples, with the samples subsequently to beexamined (at any point in time) for the existence of pathogenicmicrobial nucleic acids (bacteria, viruses etc.). I.e., reaction areasare provided which are needed for the lysis of a biological sample forthe extraction methods used according to the state of the art and thediagnostic detection of microbial nucleic acids. Preferably, these arereaction areas manifesting all the components necessary for theisolation of pathogenic nucleic acids from patient samples in a complex,storage-stable formulation:

In particular, this is a question, for the detection of microbialnucleic acids, of:

standard reaction areas entailing

-   1. at least one standard nucleic acid (control standard to verify    the extraction efficiency)-   2. at least one carrier nucleic acid (necessary in order to be able    to extract even the slightest quantities of microbial nucleic acid)-   3. a lysis buffer formulation and-   4. if need be, a proteolytic enzyme (e.g. a proteinase).

All these components are contained in the reaction cavities as mainly orcompletely water-free reagent formulations and manifest practically novolume due to their slight quantity. The production of the complex,storage-stable reagent formulations is done by specialists, preferablyeither by vacuum drying or by lyophilisation.

The reaction areas prepared in this way have numerous benefits. Inparticular, they are suited as integral parts of test kits for detectionand isolation of pathogenic nucleic acids. For example, they enable theprocessing of a complex biological sample with the objective of thequantitative detection of pathogenic nucleic acids, which starts by thetransfer of the sample into the reaction vessel according to theinvention. All the components for the lysis of the original material andextraction standards necessary for a quantitative nucleic acid diagnosisare already in the reaction vessel. In this way, no further pipettingsteps for the addition of buffers or other essential components arenecessary any more. Thus, the necessary “hands-on” efforts aredrastically reduced with a simultaneous distinct reduction of thecontamination risk. The more samples are to be processed parallel, themore important this becomes. It also becomes clear to a specialist thatautomation is made very simple by the inclusion of the reactionmaterials according to the invention.

A further essential benefit is the fact that the volume of the clinicalsample to be examined can be distinctly increased by using the reactionareas according to the invention. Thanks to the complete lack of liquidreaction components, the volume of the sample added finally alsocorresponds to the volume of the overall reaction. The multiple loadingsteps of centrifugation filters necessary up to now in an increase ofthe volume of the biological sample are no longer necessary. After thelysis of the original material, the lysate is mixed with defined shares,e.g. of an alcohol, and subsequently passed over a solid phase which isin a position to bind the nucleic acid of the sample, The solid phase isthen washed with washing buffers containing ethanol and the nucleic acidof the sample, including the extraction standard, released from thesolid phase by addition of a low-salt buffer. The nucleic acid is nowavailable for a subsequent quantitative analysis.

The test kit according to the invention entails not only the reactionareas, but also

-   -   a solid phase known to specialists for the binding of nucleic        acids,    -   if need be, binding buffers known per se,    -   if need be, washing buffers known per se,    -   if need be, low-salt buffers known per se and also, if need be,        further ancillaries also known to a specialist.

Surprisingly, it is also seen that the nucleic acid from the samplesremains stable on the solid phase for a long time after the necessarywashing steps and a subsequent drying of the solid phase. This has theadvantage that the nucleic acid in this bound form can be stored or evendispatched at ambient temperature without any problems. Complicatedlong-term storages of nucleic acid from samples at −80° C. and alsostorage with addition of ethanol are thus no longer necessary. Ifrequired, the nucleic acid from the samples is simply removed from thesolid phase by addition of a low-salt buffer and transferred todiagnostics.

Surprisingly, RNA can also be stored for such long periods without theoccurrence of degradations and can be removed from the solid phase atany required point in time. In this way, a completely new archivingsystem for nucleic acid from samples is available.

The solid phases are customarily integral parts of filtration units,which can be available as “single tube” and also as multiple variants ofindividual reaction cavities (e.g. 96-well filtration plates, 384-wellfiltration plates etc.). They are thus compatible with the reactioncavities according to the invention and permit the isolation orarchiving of nucleic acid from samples in varying formats.

A further advantage of the reaction areas used is seen in the fact thatthe nucleic acid contained is protected under the used lysis bufferformulations and added carrier nucleic acids following addition of thefluid patient sample. This permits transport of the sample withoutcooling and thus also considerably facilitates the logistics of samplecollection and sample dispatch for a subsequent quantitative diagnosis,specifically of microbial nucleic acids. Finally, it is stated that theagent according to the invention can also be used for many otherquestions of molecular diagnosis and is thus not only restricted to usein microbial diagnosis.

The application of the reaction areas according to the invention is tobe explained below in more detail with an example. Example of embodiment

ISOLATION OF VIRAL NUCLEIC ACIDS FROM BODY FLUIDS (SERUM, PLASMA, URINEETC.)

Mixing of 200 μl serum with 200 μl H₂O. Transfer of the sample to thereaction area according to the invention (2 ml reaction vessel)containing a storage-stable reagent formulation in a water-free orpredominantly water-free form, comprising:

-   1. DNA and RNA of a synthetic gene (pMS 1; Springer Lab Manual;    Quantification of mRNA by Polymerase Chain Reaction; Springer    Verlag 1995) as a standard nucleic acid for the extraction control-   2. tRNA as a carrier nucleic acid-   3. Lysis buffer containing CTAB, polyvinylpyrrolidone, ammonium    chloride, Tris-HCl, proteinase K

Mixing of the solution and incubation for 10 min at 56° C. in athermo-mixer. Subsequent addition of 400 μl isopropyl alcohol andtransfer of the solution to a centifugation column (e.g. centrifugationcolumn of the firm of Invitek with fibre glass material contained).Centrifugation for 1 min and rejection of the filtrate. Double washingof the centrifugation column with washing buffers containing ethanol.Drying of the centrifugation column by 3-minute centrifugation.

Addition of 80 μl of an elution buffers (10 mM Tris HCl; pH 8.5) to thecentrifugation column and centrifugation for 1 min.

Detection of the isolated viral RNA and/or DNA is done by means of knownamplification techniques.

1. A reaction vessel for acceptance and storage of complex biologicalsamples comprising complex, storage-stable reagent formulations in asolid form suitable for the isolating of nucleic acids.
 2. Thereaction_vessel_according to claim 1, further comprising standardreaction areas entailing at least one standard nucleic acid at least onecarrier nucleic acid a lysis buffer formulation; wherein all componentsare present in a dry or substantially water-free state.
 3. The reactionvessel according to claim 1 wherein the standard reaction areas are 1.5ml or 2 ml reaction vessels, 96-well micro-titre plates or 384-wellmicro titre plates, with the individual wells containing identical ordiffering reagents.
 4. The reaction_vessel_according to claim 1comprising nucleic acids as control standards for the verification ofthe extraction efficiency.
 5. The reaction_vessel_according to claim 1,comprising RNA, poly-RNA or DNA as carrier nucleic acids (for extractingsmall amounts of microbial nucleic acid).
 6. Thereaction_vessel_according to claim 1 further comprising detergents, monoor polyvalent cations, and at least one buffering component.
 7. Thereaction_vessel_according to claim 1, wherein they further comprise oneor more proteolytic enzymes. 8-10. (Cancel).
 11. Test kit for detectionand quantitative isolation of microbial, preferably viral and bacterialnucleic acids, comprising, reaction vessel according to one of theclaims 1 to 7 a solid phase for the binding of the nucleic acids, andoptionally one or more components selected from the group consisting ofbinding buffers washing buffers and low-salt buffers.
 12. An archivingsystem for clinically relevant nucleic acids entailing a solid phaseaccording to claim 11 manifesting the bound nucleic acid in astorage-stable form in a predominantly or completely dry state.
 13. Thereaction vessel of claim 7, wherein the proteolytic enzymes compriseproteinase K.
 14. A method for using the reaction vessel of claim 1 forisolating or detecting nucleic acids comprising: (a) providing thereaction vessel of claim 1, wherein the vessel comprises a dry nucleicacid-isolating formulation having a lysis buffer component; (b) adding abiological sample comprising nucleic acids to the reaction vessel and anamount of water effective to solubilize the dry nucleic acid-isolatingformulation; and (c) incubating the biological sample under conditionseffective for lysing the biological sample and release of the biologicalsample's nucleic acid molecules.
 15. The method of claim 14 wherein thedry nucleic acid-isolating formulation comprises a known amount ofcontrol nucleic acid for monitoring the efficiency of the isolation 16.The method of claim 14 wherein the dry nucleic acid-isolatingformulation comprises at least one proteolytic enzyme.
 17. The method ofclaim 14, wherein the biological sample's nucleic acids are viral orbacterial.
 18. The method of claim 14, wherein at least one of thebiological sample's nucleic acids are selected from the group consistingof pathogenic organisms, yeast and protozoa.
 19. The method of claim 14,wherein the biological sample's nucleic acids are microbial.